ChIP-Seq Spike-In Normalization
Compare Between ChIP-Seq Datasets with Confidence
How Quantitative is Traditional ChIP-Seq?
ChIP-Seq is a powerful tool for genome-wide identification of transcription factor binding sites and histone post-translational modifications, but its inherent semi-quantitative nature can potentially limit researchers' ability to compare differences between samples in some cases (described in greater detail in this webinar). However, by "spiking in" a small amount of exogenous chromatin into your samples prior to the ChIP reaction, you can normalize the signal from your experimental samples to this control in your final sequencing data. This approach allows researchers to be more confident than ever in the accuracy of their inter-sample comparisons.
How Can Spike-In Normalization Improve ChIP-Seq Analysis?
One particularly compelling example of the importance of ChIP-Seq normalization is illustrated in experiments investigating the genome-wide localization of the repressive histone mark H3K27me3 in the presence of epigenetic inhibitors. H3K27me3 has one identified methyltransferase (EZH2) responsible for catalyzing methyl group addition. However, EZH2 inhibition did not appear to significantly affect H3K27me3 levels in a ChIP-Seq experiment (top two tracks below).
Following normalization to spike-in, the significant loss of H3K27me3 becomes apparent (bottom two tracks below). Therefore, Spike-in can reveal changes in histone modifications that would otherwise be masked by experimental artifacts.
ChIP Normalization reveals reduction in H3K27me3 caused by an EZH2 inhibitor.
How Does ChIP-Seq Spike-In Work?
ChIP normalization can be easily implemented simply by integrating our Spike-in reagents into your standard ChIP protocol using experimental chromatin and an antibody of interest. Spike-in Chromatin and Spike-in Antibody are also added, or "spiked in", to the ChIP reaction as a minor fraction of the IP reaction. Any variation introduced during the ChIP reaction will also occur with the Spike-in chromatin. As Spike-in chromatin is consistent across all samples, a normalization factor to be created based on the Spike-in signal and applied to the sample genome.
Click on the Method tab below for a more detailed description of the Spike-in strategy.
ChIP Spike-in Normalization Advantages
- Reduce the effects of technical variation
- Detect subtle biological differences that are not observed with standard ChIP analysis
- Can be applied across different antibodies and samples without bias
- Spike-in Chromatin and Spike-in Antibody can be used with any ChIP kit or protocol
- Strategy works with both qPCR and ChIP-Seq analysis
To learn more about the ChIP Normalization Strategy, click on the Method, Data, or Contents tabs below. To view a protocol or other related documents, click on the Documents tab below.
|Spike-in Antibody||50 µg||61686||$215||Buy|
|Spike-in Chromatin||15 rxns||53083||$195||Buy|
|Drosophila Positive Control Primer Set Pbgs||96 rxns||71037||$110||Buy|
|Drosophila Negative Control Primer Set 1||96 rxns||71028||$110||Buy|
|Drosophila Negative Control Primer Set 3||96 rxns||71038||$110||Buy|
|Tech Note: Sonication Recommendations for ChIP|
|The Spike-in Normalization Strategy|
|Drosophila Negative Control Primer Set 1|
|Drosophila Negative Control Primer Set 3|
|Drosophila Positive Control Primer Set Pbgs|
How does the ChIP Spike-in Normalization Strategy work?
The diagram below depicts how easily Spike-in chromatin and a Spike-in Antibody can be integrated into an existing ChIP-Seq workflow. Spike-in Chromatin and the Spike-in Antibody are added to experimental chromatin and the experimental antibody just prior to immunoprecipitation. The Spike-in Antibody recognizes a histone variant that is specific to the species of the Spike-in chromatin (Drosophila), and the experimental antibody specifically recognizes the experimental chromatin. This enables specific detection of the Spike-in Chromatin without any significant increase in background signal. Following sequencing, reads will be mapped to their specific species. Variation introduced during the ChIP procedure will affect the Spike-in Chromatin in the same manner as the experimental chromatin, so a normalization factor can be created from the Spike-in Chromatin and applied to the experimental chromatin to normalize out technical variation and sample bias, or to monitor biological effects.
Figure 1: ChIP-Seq Normalization Workflow.
ChIP Spike-in Reaction Guidelines
Each lot of Spike-in Chromatin is quantified and tested with the Spike-in Antibody. This allows us to recommend an appropriate amount of Spike-in Chromatin to use in your reaction. A minimum amount of Spike-in Chromatin is necessary to accurately normalize the sample, but Spike-in reads should take up no more than 5% of your sequencing run. The table below is an example of the recommendations made. A lot-specific data sheet accompanies each shipment, specifying the amount of Spike-in Chromatin recommended.
|Sample Chromatin||Spike-in Chromatin||Antibody of Interest||Spike-in Antibody|
|Robust antibodies against abundant histone modifications||25 µg||Refer to lot-specific data sheet||4 µg||2 µg|
|Antibodies against transcription factors, histone modifiers or low abundance histone modifications||25 µg||Refer to lot-specific data sheet||4 µg||2 µg|
Reduce the Effects of Technical Variation
The ChIP Normalization Strategy is ideal to correct for differences that results from sample loss, amplification bias, uneven sequencing read depth or hand-to-hand differences between users. By utilizing the differences observed between samples with the Spike-in chromatin, a normalization factor is created and applied to the experimental samples to normalize out the effects of technical variation.
Figure 1: ChIP Normalization of technical variation by ChIP qPCR.
Identify Biological Differences Not Observed by Standard ChIP Analysis
By adding Spike-in Chromatin and Spike-in Antibody to standard ChIP reactions, experimental data can be normalized for sample variation. This normalization makes it easier to monitor the effects of experimental conditions, such as inhibitory compounds or mutants to reveal biological differences.
Figure 2: Normalization of biological differences in ChIP-Seq.
Specificity of Detection
The Spike-in chromatin consists of Drosophila melanogaster chromatin prepared from Schneider's Drosophila Line 2 (S2) cells. The Spike-in antibody recognizes a Drosophila-specific Histone variant, H2Av. Because of the specificity of the Spike-in antibody for the Spike-in chromatin modification, there is no cross-reactivity with mammalian samples leading to reduced background signal.
Figure 3: Specificy of the Spike-in Antibody.
Contents & Storage
Please note that the ChIP Normalization reagents are available separately. Both the Spike-in Chromatin and Spike-in Antibody are required to apply the normalization strategy. Drosophila-specific qPCR primer sets are available for ChIP qPCR analysis.
- 50 µg Spike-in Antibody supplied at a concentration of 1 µg/µl in PBS containing 0.035% sodium azide and 30% glycerol. Spike-in Antibody is shipped at room temperature. This will not affect the stability or performance of the reagent. Upon receipt, store at -20°C. Avoid repeated freeze/thaw cycles.
- Spike-in Chromatin prepared from Schneider's Drosophila Line 2 (S2) cells is provided for 15 rxns of robust histone modification antibody targets. Spike-in chromatin is provided at a concentration of 10 ng/µl.
qPCR Primer Sets
- 400 µl Drosophila Positive Control Primer Set Pbgs is supplied at a concentration of 2.5 µM
- 400 µl Drosophila Negative Control Primer Set 1 is supplied at a concentration of 2.5 µM